U.S. patent application number 13/819989 was filed with the patent office on 2013-06-27 for semi-stable production of lentiviral vectors.
This patent application is currently assigned to Via Olgettina, 58. The applicant listed for this patent is Chiara Bovolenta, Fulvio Mavilio, Paolo Rizzardi, Anna Stornaiuolo. Invention is credited to Chiara Bovolenta, Fulvio Mavilio, Paolo Rizzardi, Anna Stornaiuolo.
Application Number | 20130164840 13/819989 |
Document ID | / |
Family ID | 49582836 |
Filed Date | 2013-06-27 |
United States Patent
Application |
20130164840 |
Kind Code |
A1 |
Bovolenta; Chiara ; et
al. |
June 27, 2013 |
Semi-Stable Production of Lentiviral Vectors
Abstract
The present invention provides a new semi-stable packaging cell
line and a method to produce lentiviral vectors (LV), using the
semi-stable packaging cell line. New methods and packaging cell
lines of the invention are generated using a baculo-AAV hybrid
system for stable expression of structural and regulatory
lentiviral proteins, such system comprising a baculoviral backbone
containing an integration cassette flanked by AAV ITRs, in
combination with a plasmid encoding rep protein. This system allows
to obtain a stable integration of the structural and regulatory
HIV-1 proteins gag/pol and rev. The system allows to obtain a cell
line including the structural and regulatory HIV proteins gag/pol
and rev, to be used for a semi-stable LV production.
Inventors: |
Bovolenta; Chiara; (Milan,
IT) ; Stornaiuolo; Anna; (Milan, IT) ;
Rizzardi; Paolo; (Milan, IT) ; Mavilio; Fulvio;
(Evry, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bovolenta; Chiara
Stornaiuolo; Anna
Rizzardi; Paolo
Mavilio; Fulvio |
Milan
Milan
Milan
Evry |
|
IT
IT
IT
FR |
|
|
Assignee: |
Via Olgettina, 58
Milan
IL
|
Family ID: |
49582836 |
Appl. No.: |
13/819989 |
Filed: |
September 1, 2011 |
PCT Filed: |
September 1, 2011 |
PCT NO: |
PCT/EP2011/065089 |
371 Date: |
February 28, 2013 |
Current U.S.
Class: |
435/367 ;
435/320.1; 435/366; 435/369 |
Current CPC
Class: |
C12N 2800/40 20130101;
C12N 2710/14144 20130101; C12N 2750/14143 20130101; C12N 15/86
20130101; C12N 15/8673 20130101; C12N 2740/16052 20130101; C12N
2800/50 20130101 |
Class at
Publication: |
435/367 ;
435/320.1; 435/366; 435/369 |
International
Class: |
C12N 15/867 20060101
C12N015/867 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 2, 2010 |
EP |
10175088.3 |
Claims
1. A system for stable expression of lentiviral structural and
regulatory proteins consisting of: i. a hybrid vector comprising
baculoviral backbone containing an integration cassette flanked by
adeno-associated virus (AAV) inverted terminal repeats (ITRs)
including two expression cassettes, wherein the first expression
cassette encodes lentiviral gag and pol and the second cassette
encodes lentiviral rev and a selection marker; and ii. an
expression plasmid containing the AAV Rep Open Reading Frame (ORF)
under the control of a promoter.
2. The system according to claim 1 wherein the two expression
cassettes of the hybrid vector are tail to tail oriented and each
one is driven by a constitutive promoter and a poly A.
3. The system according to claim 1 wherein the promoter is selected
from cytomegalovirus (CMV), CMV immediate-early (IE),
phosphoglyeerate kinase (PGK), simian virus 40 (SV40), elongation
factor 1 alpha (eF1.alpha.), spleen focus-forming virus (SFFV), and
rous sarcoma virus (RSV).
4. The system according to claim 1 wherein the promoter is a CMV IE
promoter,
5. The system according to claim 1 wherein the selection marker is
selected from a hygromycin, kanamycin, neomycin, and zeomycin
resistance gene.
6. The system according to claim 5 wherein the selection marker is
a hygromycin resistance gene.
7. The system according to claim 1 wherein the selection marker is
cloned downstream from an internal ribosome entry site (IRES).
8. The system according to claim 1 wherein the rep protein is
rep78.
9. A semi-stable lentiviral packaging cell line consisting of cells
stably expressing lentiviral gag, pol and rev characterized in that
such cells contain stably integrated into its genome at least one
copy of an integration cassette flanked by AAV inverted terminal
repeats (ITR) including two expression cassettes, wherein the first
expression cassette encodes lentiviral gag and pol and the second
cassette encodes lentiviral rev and a selection marker.
10. The semi-stable lentiviral packaging cell line according to
claim 9 wherein the cell is a human cell line selected from HEK293,
HEK293-T, HEK293-SF, TE671, HT1080 and HeLa.
11. The semi-stable lentiviral packaging cell line according to
claim 9 wherein the two expression cassettes are tail to tail
oriented and each one is driven by a constitutive promoter and a
poly A.
12. The semi-stable packaging cell line according to claim 9
wherein the promoter is selected from CMV, CMV IE, PGK, SV40,
eF1.alpha., SFFV and RSV.
13. The semi-stable packaging cell line according to claim 12
wherein the promoter is a CMV IE promoter.
14. The semi-stable packaging cell line according to claim 9
wherein the selection mailer is selected from a hygromycin,
kanamycin, neomycin, and zeomycin resistance gene.
15. The semi-stable packaging cell line according to claim 14
wherein the selection marker is a hygromycin resistance gene.
16. The semi-stable packaging cell line according to claim 9
wherein selection marker is cloned downstream an internal ribosome
entry site (IRES).
17. A method for producing lentiviral vectors comprising: i.
culturing a semi-stable packaging cell line according to claim 9;
ii. inserting in the semi-stable packaging cell line an env gene;
and iii. inserting in the semi-stable packaging cell line a
transfer vector.
18. The method according to claim 17 wherein the env gene is
selected from vesicular stomatitis virus-glycoprotein (VSV-G) env,
murine leukemia virus (MLV) 4070 env, endogenous feline virus RD114
env, chimeric envelope protein RD114-TR, chimeric envelope protein
RD114-pro, baculovirus GP64 env, and GALV env or derivatives
thereof.
19. The method according to claim 17 wherein the env gene is the
gene encoding the chimeric envelope protein RD 114-TR.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the production of
lentiviral vectors (LV) for gene therapy. More particularly, the
invention relates to semi-stable lentiviral packaging cell lines
and methods of manufacturing packaging cell lines using an hybrid
baculo-Adeno associated virus (AAV) vector for stable integration
of structural and regulatory lentiviral proteins.
BACKGROUND
[0002] Since the first-ever LV phase I clinical trial against AIDS
in 2001, 38 phase I-II and two phase II-III trials exploiting
HIV-based LV as gene delivery vehicles have undergone authorities'
scrutiny; three of them are still under review. The largest number
of trials comprises monogenic disorders, some of which with large
incidence such as Cooley's anemia .beta.-thalassemia major (4
trials). Cancer and infectious diseases, mostly HIV-1 infection,
follow. Commonly, the number of patients enrolled in phase I/II
trials is limited, but not that in phase III. Thus stable packaging
cell lines for 2.sup.nd (LTR-based) and 3.sup.rd (SIN-based) LV
generation are urgently needed to cope LV large scale production
demand for phase III trials hopefully attainable in the future in a
larger number. LV production grounded on transient protocols is
indeed impractical for global application under a safety, cost and
reproducibility standpoint.
[0003] An increasing body of evidence indicates that LV, the most
recently developed viral integrating vectors for gene therapy, are
broadly applicable to transduce either terminally differentiated or
cycling cells, ideal to sustain long-term transgene expression and
safer than what was initially feared. The experience accumulated on
Moloney murine leukemia virus (MoMLV) gamma-retroviral vectors
(.gamma.RV) over the last two decades guided the fast progress on
LV delivery system, whose development originated by the necessity
of overcoming the inability of retrovirus to transduce non diving
cells. In particular, the generation of self-inactivating (SIN)
transfer vectors makes the prospect of a large use of LV in human
clinical trials more feasible.sup.1 provided the expansion and
optimization of an as much efficient manufacturing process.
However, in contrast to .gamma.RV, which can be produced by several
human and murine commercially available packaging cell lines, LV
are currently produced not only for research-grade, but also for
GMP-grade, almost exclusively by transient transfection. This
technology is expensive, difficult to standardize and scale-up and
requires numerous downstream validation tests. Furthermore, the
risk of replication competent lentiviruses (RCL), possibly arising
through recombination between viral sequences in the packaging and
transfer vector constructs, is a rare, but more likely event during
transient than stable production.
[0004] The development of a retroviral-equivalent stable packaging
cell line for LV turned out to be slower and more difficult
because, as opposite to gamma retrovirus, the expression of
lentiviral proteins, such as the env, protease, and some accessory
proteins is toxic for human cells. To overcome this problem the
accessory genes, present in the very early versions of packaging
cells, were later removed in the latest generations. First
generation SIV- and HIV-based LV packaging cell lines were obtained
from either monkey Vero, or human COS, HeLa and 293 adherent
cells.sup.2-5, engineered with lentivirus genomes carrying few
crucial modifications such as the removal of the packaging signal.
The gp120 env and most accessory genes were in fact maintained. The
resulting LV titer was very low.sup.2-5, and more importantly the
possible application of these vectors was necessarily restricted to
CD4.sup.+ T cells for anti-AIDS gene therapy approaches. Later,
gp120 env was substituted with the glycoprotein derived from the
vesicular stomatitis virus (VSV-G) and all accessory genes were
removed because proven dispensable for an efficient LV production.
To prevent the toxicity also described for VSV-G, its expression
was conditionally induced by a variety of different systems, such
as the Tet, ecdysone, Rev and the combination of Tet and
cumate.sup.6. Similarly, to reduce the toxic effect of the viral
protease during clone selection, the conditional expression of the
gag-pol gene by the Tet and the combination of doxycycline and
cumate drugs have been described.sup.6. In all these systems
gag-pol, rev and env genes were integrated by transient
transfection of plasmid DNA, followed by drug selection and cell
cloning.
[0005] One of the crucial issue for the implementation of a truly
stable packaging cell line is the choice of the best viral gene
delivery vehicles. Most researchers integrated the gag-pol, rev and
env genes by transient transfection of plasmid DNA, followed by
drug selection and cell cloning.sup.2-9. This technology is known
to suffer over time from gene silencing and gene loss.sup.10, which
can both jeopardize the long-term stability of the packaging
clone.
[0006] Alternative gene delivery vehicles have been disclosed
particularly in STAR.sup.11 and in the more recently developed
GPRG-TL-20.sup.12 packaging cell lines where the gag, pol, and rev
genes were integrated into HEK293T cells by MLV-shuttle vectors.
Two copies of the recoded gag-pol gene were stably integrated in
STAR, whereas no such information is available for
GPRG-TL-20.sup.12. As opposite to STAR, where the env gene were
transfected, in GPRG-TL-20 all the remaining viral genes were
introduced by SIN-MLV.
[0007] Several systems exist that allow stable integration of
foreign genome into host cells. Palombo et al., 1998.sup.13
disclose an hybrid baculovirus-AAV vector for specific integration
into host cells. Such vector appears to be very effective if it
includes rep gene in the same hybrid baculovirus-AVV vector. There
is no mention in this reference of the construct of the present
invention let alone the suggestion of using this kind of system for
LV production.
[0008] Over the last almost two decades, several attempts to
generate stable LV packaging cell lines have been made. Despite the
different technology disclosed, as of today none of these packaging
cell lines is employed in clinical trials or corners the market
yet. Therefore there is a need of new systems for large scale
production of LV that are effective in terms of production
capability and are safe, cost effective and reproducible.
SUMMARY OF THE INVENTION
[0009] The present invention is related to the field of production
of LV. Several gene therapy clinical trials are ongoing employing
LV as gene delivery vehicles. In all these trials LV production is
still based on transient protocols.
[0010] The present invention provides a new strategy to generate an
HIV-1-based packaging cell line. Such strategy is based on the use
of a hybrid vector comprising baculoviral backbone containing an
integration cassette flanked by AAV inverted terminal repeats
(ITRs), the so-called baculo-AAV hybrid system, in combination with
a plasmid encoding a rep protein. This system allows to obtain a
stable integration of structural and regulatory HIV-1 proteins
gag/pol and rev. The system of the present invention includes a) a
hacuio-AAV hybrid vector characterized in that it contains two
expression cassettes, one encoding lentiviral gag and pol genes and
the other lentiviral rev and a selection marker, and b) a plasmid
encoding a rep protein. The proposed system represents a new and
advantageous way to deliver structural and regulatory HIV proteins
in order to stably and effectively engineer host cells with
structural lentiviral proteins. Using this system, it was obtained
a first intermediate including only structural and regulatory HIV
proteins gag/pol and rev, to be used for a semi-stable LV
production, or as starting point to obtain 2.sup.nd and 3.sup.rd
generation packaging cell lines including optionally the regulatory
protein (Tat) and the envelope protein of interest, as well as
producer cell lines including also the transfer vector.
[0011] The first intermediate carries two copies of the recombinant
baculo-AAV packaging construct expressing the HIV-1 gag-pol and rev
genes in a tri-cistronic configuration. Such intermediate has been
called PK-7 and is referred to as PK-7 in the examples. Genome
integration of baculo-AAV packaging vector was facilitated by the
transient expression of the AAV Rep78 protein known to be necessary
for an ITR-mediated AAV vector integration.sup.14. Such first
intermediate showed to have a surprising genetic stability for 1
year of culture that has proven the continuous production of
functional LV after transient transfection of the remaining genetic
elements. In addition, no silencing phenomenon have been observed
in such cells. Furthermore, by exactly mapping the integration site
of the two tandernly integrated packaging AAV vectors in a
non-coding intergenic transcriptionally active region, we have
provided a safety argument against the possible activation of
dangerous genes whose mRNA can be incorporated in the LV and,
eventually, in the host target cells.
[0012] The developed packaging system based on the use of an hybrid
baculo-AAV vector for stable expression of lentiviral gag-pol and
rev has been called "MolPack". The expression system used in the
present invention advantageously allows stable and safe
introduction of structural (gag/pol) and regulatory (rev) HIV
proteins, in only one transfection and cloning round. The
production of LV currently employed in clinical trials is still
based on transient transfection of all required proteins. On the
contrary, the intermediate obtained with the expression system of
the present invention is stable, does not show silencing phenomena,
allows to develop a semi-stable production of LV with strong
advantages from the economic point of view. In fact, the
semi-stable production requires a reduced number of constructs to
be transfected to obtain the final product. In addition, it has
also remarkably found that, using the semi-stable production of the
present invention, only one third of the DNA amount employed in the
production with transient transfection is sufficient to obtain LV
having a titer comparable to that of transiently produced LV.
Moreover, the reduced number of constructs to be transfected
reduces the possibility of recombination events with the formation
of RCL, thus rendering lentiviral particle production safer.
Therefore, the expression system, the semi-stable packaging cell
line and the production method of the present invention are very
advantageous in respect to tools and methods currently applied in
clinical trials.
Statements of the Invention
[0013] According to a first aspect of the invention there is
provided a system for stable expression of lentiviral structural
and regulatory proteins consisting of: [0014] i. An hybrid vector
comprising baculoviral backbone containing an integration cassette
flanked by AAV ITRs including two expression cassettes, wherein the
first expression cassette encodes lentiviral gag and pol genes and
the second one lentiviral rev and a selection marker and [0015] ii.
An expression plasmid containing the AAV rep Open Reading Frame
(ORF) under control of a promoter.
[0016] Preferably the two expression cassettes of the hybrid vector
are tail-to-tail oriented and each one is driven by a constitutive
promoter and a poly A, preferably the promoter is selected from
CMV, CMV IE, PGK, SV40, eF1.alpha. SFFV and RSV more preferably the
promoter is a CMV IE promoter.
[0017] Preferably the selection marker is selected from hygromycin,
kanamycin, neomycin, zeomycin resistance gene, more preferably the
selection marker is hygromycin resistance gene.
[0018] More preferably the selection marker is cloned downstream an
internal ribosome entry site (IRES). According to another aspect of
the invention, there is provided a semi-stable lentiviral packaging
cell line consisting of cells stably expressing lentiviral gag pol
and rev characterized in that such cells contain stably integrated
into its genome at least one copy of an integration cassette
flanked by AAV ITRs including two expression cassettes, wherein the
first expression cassette encodes lentiviral gag and pol genes and
the second one lentiviral rev and a selection marker.
[0019] Preferably the cell is a human cell line preferably selected
from HEK293, HEK293-T, HEK293-SF, TE671, HT1080 or HeLa, more
preferably the cell line is HEK293-T.
[0020] Preferably the two expression cassettes are tail-to-tail
oriented and each one is driven by a constitutive promoter and a
poly A; preferably the promoter is selected from CMV, CMV IE, PGK,
SV40, eF1.alpha., SFFV and RSV, more preferably the constitutive
promoter is a CMV IE promoter.
[0021] Preferably the selection marker is selected from hygromycin,
kanamycin, neomycin, zeomycin resistance gene; more preferably the
selection marker is hygromycin resistance gene. More preferably the
selection marker is cloned downstream an IRES.
[0022] Preferably the AAV rep protein is selected from rep78 or
rep68. More preferably rep protein is rep78. According to another
aspect there is provided a method for producing lentiviral vectors
comprising: [0023] i. Culturing a semi-stable lentiviral packaging
cell line consisting of cells stably expressing lentiviral gag, pol
and rev characterized in that such cells contains, stably
integrated into its genome, at least one copy of an integration
cassette flanked by AAV ITRs including two expression cassettes,
wherein the first expression cassette encodes lentiviral gag and
pol genes and the second one lentiviral rev and a selection marker
[0024] ii. Inserting in the semi-stable packaging cell line an env
gene [0025] iii. Inserting in the semi-stable packaging cell line a
transfer vector
[0026] Preferably the two expression cassettes are tail-to-tail
oriented and each one is driven by a constitutive promoter and a
poly A; preferably the promoter is selected from CMV, CMV IE, PGK,
SV40, eF1.alpha., SFFV and RSV, more preferably the constitutive
promoter is a CMV IE promoter.
[0027] Preferably the selection marker is selected from hygromycin,
kanamycin, neomycin, zeomycin resistance gene; more preferably the
selection marker is hygromycin resistance gene. More preferably the
selection marker is cloned downstream an IRES.
[0028] Envelope protein can be inserted in host cells using AAV
vector, retroviral vector, stable plasmid integration or homologous
recombination. Preferably the env gene is inserted by transient
transfection using a plasmid.
[0029] Preferably the env gene is selected from VSV-G env, MLV 4070
env, RD114 env, chimeric envelope protein RD114-TR, chimeric
envelope protein RD114pro, baculovirus GP64 env or GALV env or
derivatives thereof, more preferably the env gene is the gene
encoding the RD114-TR.
[0030] Preferably the transfer vector is inserted with a SIN
lentiviral vector.
DETAILED DESCRIPTION OF THE INVENTION
[0031] A detailed description of preferred features and embodiments
of the invention will be described by way of non-limiting
example.
[0032] The invention can be put into practice by a person of
ordinary skill in the art who will employ, unless otherwise
indicated, conventional techniques of chemistry, molecular biology,
microbiology, recombinant DNA and immunology. All such techniques
are disclosed and explained in published literature. See, for
example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,
Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,
Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995
and periodic supplements; Current Protocols in Molecular Biology,
ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); Current
Protocols in Immunology, ch. 12, John Wiley & Sons, New York,
N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and
Sequencing: Essential Techniques, John Wiley & Sons; J. M.
Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles
and Practice; Oxford University Press; M. J. Gait (Editor), 1984,
Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D.
M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA
Structure Part A: Synthesis and Physical Analysis of DNA Methods in
Enzymology, Academic Press. All these publications are incorporated
by reference.
Baculo-AAV Hybrid System
[0033] The present invention provides a new strategy to generate an
HIV-1-based packaging cell line. Optimization of production system
for LV is one of the critical issues that needs to be solved for
the development of gene therapy medicine based on LV technology.
Despite the growing number of clinical trials employing this
technology, LV are still produced, in such trials, using transient
transfection protocols. In this way, production of LV is still very
expensive and unsatisfactory for larger number of patients. For
this reason, many efforts have been made to develop stable
packaging cell lines for LV. One of the critical issues in the
development of a stable lentiviral packaging cell line is choosing
the right vehicle for engineering host cells. In many cases host
cells have been engineered using plasmids, but, in such cases,
genome instability and gene silencing phenomena have also been
observed. Retroviral vectors have been used to stably integrate
gag/pol and rev genes in two other cases. None of the stable
packaging cell line developed so far has been employed in
clinic.
[0034] The strategy of the present invention is based on the use of
a system for stable expression of lentiviral structural and
regulatory HIV proteins consisting of an hybrid vector comprising a
baculoviral backbone containing an integration cassette flanked by
AAV ITRs including two expression cassettes, wherein the first
expression cassette encodes lentiviral gag and pol genes and the
second one lentiviral rev and a selection marker; together with an
expression plasmid containing the AAV rep ORF under control of a
promoter. The presence of baculoviral backbone allows to host a big
and complex integration cassette including two expression cassettes
encoding several different proteins. The resulting baculo-AAV
packaging vector allows to engineer host cells with viral proteins
that are necessary to stably and effectively produce LV, through
only one infection event.
[0035] Genome integration of baculo-AAV packaging vector was
obtained by the transient expression of the AAV rep protein. This
system allowed to obtain integration of AAV vectors in a non-coding
intergenic transcriptionally active region, thus excluding
activation of dangerous genes whose mRNA can be incorporated in the
LV and eventually in the host target cells.
[0036] The proposed system represents a new and advantageous way to
stably and effectively engineer host cell with structural and
regulatory lentiviral proteins.
[0037] In a preferred embodiment, the two expression cassettes
included in the baculo-AAV packaging construct are tail-to-tail
oriented and each one is driven by a constitutive promoter and a
poly A, preferably the promoter is selected from CMV, CMV IE, PGK,
SV40, eF1.alpha., SFFV, and RSV, more preferably the promoter is a
CMV IE promoter. According to a preferred aspect of the invention,
the selection marker included in the AAV packaging vector is
selected from hygromycin, kanamycin, neomycin, zeomycin resistance
gene; preferably the marker is hygromycin resistance gene; more
preferably, the selection marker is cloned downstream an IRES.
[0038] Genome integration of baculo-AAV packaging vector was
obtained by the transient expression of AAV Rep protein for an
ITR-mediated AAV vector integration. In a preferred embodiment rep
protein is selected from rep78 and rep68, more preferably the
protein is rep78.
[0039] Using this system, it was possible to obtain cells
engineered to stably express HIV-1 proteins gag/pol and rev, such
cells are called semi-stable packaging cell line. Particulariy, the
present invention discloses and claims such engineered cells and
their use for a semi-stable LV production.
Semi-Stable Packaging Cell Line
[0040] Semi-stable packaging cell line of the present invention
consists of host cells carrying at least one copy of the
recombinant baculo-AAV packaging construct expressing the HIV-1
gag-pol and rev genes. Genome integration of baculo-AAV packaging
vector has been obtained by the transient expression of the AAV rep
protein in order to obtain ITR-mediated AAV vector integration.
Preferably the two expression cassettes are tail-to-tail oriented
and each one is driven by a constitutive promoter and a poly A,
preferably the promoter is selected from CMV, CMV IE, PGK, SV40,
eF1.alpha., SFFV and RSV. More preferably the constitutive promoter
is a CMV IE promoter.
[0041] According to a preferred aspect of the invention the
selection marker is selected from hygromycin, kanamycin, neomycin,
zeomycin resistance gene; preferably the selection marker is
hygromycin resistance gene, more preferably the selection marker is
cloned downstream an IRES.
[0042] Preferably the AAV rep protein is selected from rep78 and
rep68. More preferably rep protein is rep78. Host cell lines that
can be engineered to obtain the semi-stable packaging cell line are
human cell lines selected from HEK293, HEK293-T, HEK293-SF, TE671,
HT1080 or HeLa, more preferably the cell line is HEK293-T.
[0043] Such semi-stable packaging cell line is suitable for the
output of a potentially large variety of LV, with different env and
different transfer vectors in a semi-stable production system.
Therefore, it represents a great advantage for a more effective
production of LV since it allows costs reduction, it does not
require using the GMP-grade plasmid DNA encoding gag-pol and rev,
and the risk of RCL formation secondary to recombination events
between the plasmids during transient transfection is reduced. The
semi-stable packaging cell line is a versatile tool, and is safer
and economically more advantageous than other tools currently
employed in clinical trials for the production of LV.
[0044] Semi-stable packaging cell line of the present invention
showed to have a surprising genetic stability for 1 year of culture
that has proven the continuous production of functional LV after
transient transfection of the remaining genetic elements. In
addition, no silencing phenomenon have been observed in fact, both
titer and infectivity of LV obtained using this intermediate
remained unaffected after 1 year. Such data were confirmed both in
the presence or absence of selective pressure. Remarkably, no
comparable data regarding the integration stability of an AAV-ITR
mediated cassette are available in the literature. The only related
information is that a human bone marrow derived, fibroblast-like
cell line (Ruddle's Detroit 6 cells) infected with wild type AAV
serotype 2 (AVV-2) maintained viral sequences in a latent state for
at least 47 and 118 passages. As shown in the examples the
semi-stable packaging cell line of the present invention survived
for at least 102 passages,
[0045] Therefore, the present invention provides a method for
producing LV comprising: [0046] i. Culturing a semi-stable
packaging cell line as described above [0047] ii. Inserting in the
semi-stable packaging cell line an env gene [0048] iii. Inserting
in the semi-stable packaging cell line a transfer vector
[0049] Envelope protein can be inserted in host cells using AAV
vector, retroviral vector, stable plasmid integration or homologous
recombination. Preferably the env gene is inserted by transient
transfection using a plasmid.
[0050] Preferably the env gene is selected from VSV-G env, MLV 4070
env, RD114 env, chimeric envelope protein RD114-TR, chimeric
envelope protein RD114pro, baculovirus GP64 env or GALV env or
derivatives thereof. More preferably the present invention makes
use the chimeric envelope protein RD114-TR env in which the
cytoplasmic tail of RD114 has been replaced by that of MLV
envelope.
[0051] Preferably the transfer vector is inserted with a
SIN-LV.
[0052] Semi-stable packaging cell line of the present invention are
able to produce LV that in terms of infectivity are "qualitatively"
equal to those produced by transient transfection. A very important
issue to be considered for the application of the semi-stable
production of LV is that semi-stable packaging cell line did not
lose the ability to be easily transfectable. In fact, PK-7 cells
are transfected at the same level of the parental cell line
HEK293T. This feature is quite commonly lost after genetic
modification and cloning of HEK293 cells with packaging construct.
On the contrary, the expression system employed in the present
invention for stable integration of gag/pol and rev does not cause
any problem to the transfectability of semi-stable packaging cell
line.
[0053] Moreover, the semi-stable packaging cell line and its use in
the production method for LV have important advantages for the
production costs. Particularly, only three additional constructs,
for example plasmids, (each encoding tat, env and transfer vector)
are necessary for production of 2.sup.nd generation LV, or two
constructs (each encoding env and transfer vector) for production
of 3.sup.rd generation LV. On the contrary, GMP methods currently
employed in clinical trials require the use of two additional
constructs, encoding gag/pol and rev respectively, in addition to
those used with the semi-stable packaging cell line of the present
invention.
[0054] Further, the reduced number of constructs to be transfected
with the methods and tools of the present inventions, also
represents an additional advantage for the safety of the LV as
described above.
[0055] Moreover, it was also notably found that, using the
semi-stable production of the present invention, only one third of
the total amount of DNA employed in the production by transient
transfection is sufficient to obtain lentiviruses having a titer
comparable to that of transiently produced lentiviruses. Therefore,
the expression system, the semi-stable packaging cell line and the
production method of the present invention are very advantageous in
respect to tools and methods currently applied in the clinic.
DESCRIPTION OF THE FIGURES
[0056] FIG. 1 Schemes of RD-MolPack development. (a) Schematic
representation of the DNA plasmids used in the study. pPolh,
polyhedrin promoter; attB1, attachement B1; ITR, inverted terminal
repeat; CMV, cytomegalovirus promoter; In, intron; RRE, rev
responsive element; A, polyA sequence; IRES, internal ribosome
entry site; SD, splice donor; SA, splice acceptor; .PSI., packaging
signal; WPRE, woodchuck hepatitis post-transcriptional regulatory
element; cPPT, central polypurine tract; hPGK, human
posphoglycerate kinase promoter. (b) Cartoon of the Rep78-mediated
genomic integration of the AAV-GPR vector. MV Rep78 promotes the
excision of the ITR-flanked AAV-GPR cassette and facilitates its
integration into human chromosomes. (c) Flow chart of the
production by semi-stable packaging cell line. GOI, gene of
interest.
[0057] FIG. 2 Characterization of the PK clones. (a) Southern blot
analysis of the AAV-GPR vector integration. To establish the number
of copies and the integrity of the cassette, genomic DNA (10 .mu.g)
derived from PK clones was digested with two different restriction
enzymes, BamHI and SnaBI, respectively. (b) Western blot analysis
of the viral proteins produced from the GPR cassette. Left panel,
cell extracts (50 .mu.g/lane) obtained from the PK clones were
hybridized to an anti-HIV human serum recognizing HIV-1 proteins.
The membrane was sequentially hybridized with an anti-rev specific
Ab. Right panel, 30 ng p24gag-equivalent of viral like particles
(VLP) produced from PK clones were processed identically to the
cellular extracts. (c) Schematic mapping of the GPR-cassette
integration by LM-PCR technique which identified the DNA
break-point in the long arm of chromosome 2 at the 2q32.1 location.
(d) Co-localization of the AAV-GPR cassette and the human Hox4 gene
into chromosome 2. In situ hybridization of PK-7 metaphase
chromosomes was carried out using a gag-specific (red) and a
Hox4-specific (green) probe, respectively. (e) Schematic
representation of the rearrangement of the two GPR integrated
cassettes in the PK-7 clone and their tail-to-head orientation.
[0058] FIG. 3 Control experiments relative to the Baculo-AAV-GPR
and Rep78 possible integration. (a) Southern blot analysis of the
recombinant baculovirus-AAV DNA. DNA was extracted from baculovirus
particles, digested overnight with MluI restriction enzyme and,
after blotting, probed with the 11-kb GPR cassette specific probe.
Entry-GPR plasmid (1 pg) and baculovirus empty DNA (100 ng) were
loaded as positive and negative control, respectively. (b)
Detection of putative residual rep78 plasmid DNA integration into
PK-7 cells. Rep78 specific PCR was carried out using the PK-7
genomic DNA as sample template and the CMV-rep78 (1 pg) plasmid as
positive control.
EXAMPLES
Example I
General Methods
Plasmids
[0059] Wild-type HIV-1 gag, pol and rev genes were excised by
MluI/NarI and MluI/NotI digestions from the pCG719-pKLgagpol
(hereafter named CMV-GPR for simplicity) (FIG. 1a, scheme 9) and
pCG720-pKrev (CMV-Rev) (FIG. 1a, scheme 5) plasmids,
respectively.sup.25. The viral genes were inserted into the
Gateway.RTM. pENTR.TM.4 shuttle vector (Invitrogen, Co., Carlsbad,
Calif.) in two distinct expression cassettes tail-to-tail oriented,
each cassette driven by a CMV IE promoter and carrying a polyA
sequence. The first cassette expresses the gag and pol genes
whereas the second one the rev gene and the selection marker
hygromycin resistance (hygro) gene; hygro was cloned downstream an
IRES to allow bi-cistronic translation. The two expression units
were introduced into the XbaI site of the recombinant pSUB201
plasmid carrying an infectious AAV genome.sup.17. The resulting
5'ITR-CMV-gagpol-polyA-polyA-hygro-IRES-rev-CMV-ITR3' cassette was
then excised and inserted into the Gateway.RTM.pENTR.TM.4 shuttle
vector (Invitrogen, Co., Carlsbad, Calif.). The recombinant hybrid
baculo-AAV packaging vector (Baculo-AAV-GPR) (FIG. 1a, scheme 1)
was obtained by means of the bacteriophage lambda site-specific
recombination system between the pENTR.TM.4 shuttle entry vector,
containing the two cassettes, and the BaculoDirect Linear DNA
(BaculoDirect.TM. Baculovirus Expression Systems, Invitrogen, Co.).
During homologous recombination the polyhedrin gene of the baculo
DNA was thereby replaced with the GPR double cassette. The
pABCMV-rep78 expression plasmid (CMV-AAV-rep78) was obtained by
cloning the AAV-rep78 ORF under the CMV IE promoter of the
expression vector pABS.43 as described in Recchia et al.,
2004.sup.18 (FIG. 1a, scheme 4). The pMD.G plasmid
(CMV-VSV-G).sup.19, encodes the vesicular stomatitis envelope
glycoprotein (VSV-G) (FIG. 1a, scheme 6). The 3.sup.rd-generation
transfer vector, pCCLsin.PPT.hPGK.eGFP.WPRE.Amp (SIN-eGFP).sup.20
expresses the eGFP gene under the constitutive promoter hPGK (FIG.
1a, scheme 2). The 2.sup.nd-generation P.DELTA.N-Chim3 transfer
vector expressing the anti-HIV-1 Chim3 transgene was described in
Porcellini et al., 2009 & 2010.sup.21,22 (FIG. 1a, scheme 3).
The pCMV-RD114-TR (CMV-RD114-TR) (FIG. 1a, scheme 7) plasmid
encodes the chimeric RD114-TR envelope, made of the extracellular
and trans-membrane domains of the feline endogenous retrovirus
RD114 envelope and the cytoplasmic tail (TR) of the A-MLVenv
4070A.sup.23. The 2.sup.nd-generation packaging pCMV-.DELTA.R8.74
(CMV-GPRT) construct (FIG. 1a, scheme 8) encoding the HIV-1 gag,
pol, rev and tat genes.sup.19.
Cells
[0060] Spodoptera frugiperda (Sf9) insect cells (Invitrogen, Co.)
were grown in suspension in TC-100 medium (Invitrogen, Co.)
supplemented with 10% FCS (EuroClone Ltd, UK) and a combination of
penicillin-streptomycin and glutamine (PSG) at 27.degree. C. in the
absence of CO.sub.2. Human embryo kidney 293T (HEK293T) cells and
its derivative clone (PK-7) were propagated in Dulbecco's Modified
Eagle Medium (DMEM) supplemented with 10% FCS and PSG. CEM A3.01
and SupT1 T lymphoblastoid cells were grown in RPMI 1640
supplemented with 10% FCS and PSG. CD34.sup.+ haemopoietic stem
cells (HSC) and neonatal leukocytes were purified from umbilical
cord blood (UCB) centrifugation on a Ficoll-Hypaque gradient
(Lymphoprep, Nycomed Pharma AS, Oslo, Norway). After gradient
separation, CD34.sup.+ HSC were isolated from the collected UCB
mononucleated cell ring by positive selection using CD34 MicroBeads
Kit and MiniMACS Separator Columns (Miltenyi Biotec, Sunnyvale,
Calif.). CD34.sup.+ cells purity (>92%) was established by FACS
analysis (FACSCalibur BD Bioscience, San Jose, Calif.) and the
FlowJo software (Tree Star, Inc., Ashland, Oreg.), using the
anti-CD34-PE Ab (BD Pharmingen.TM., San Diego, Calif.). CD34.sup.+
cells were pre-stimulated for 24 hours in 20% serum Iscove's
Modified Dulbecco's Medium (IMDM) containing human stem cell factor
(h-SCF) 100 ng/ml (R&D Systems, Minneapolis, Minn.), h-FIt3L
100 ng/ml (Peprotech, Rocky Hill, N.J.), h-IL-6 20 ng/ml (R&D
Systems) and human thrombopoietin (h-Tpo) 20 ng/ml (Peprotech) and
maintained in the same medium during transduction. Neonatal
leukocytes were stimulated for 48 hours with the soluble anti-human
CD3 (30 ng/ml) (Orthoclone OKT3, Janssen-Cilag, UK) and recombinant
human IL-2 (rhIL-2) 50 U/ml (Chiron, Emeryville, Calif.) in RPMI
and then kept in RPMI supplemented with 10% FCS, PSG, and
rhIL-2.
Baculovirus Production and Baculo-GPIR Infection of HEK293T
Cells
[0061] Baculovirus, carrying the recombinant hybrid Baculo-AAV-GPR
DNA genome, was produced following the BaculoDirect method using
the Gateway.RTM. adapted Baculovirus DNA system (Invitrogen, Co.).
Recombinant Baculovirus titer was evaluated by plaque assay and
corresponded to 1.times.10.sup.11 pfu/ml after three passages of
viral amplification in Sf9 cells. PK-7 clone was obtained by
transfecting 1.5.times.10.sup.6 HEK293T cells with 4 .mu.g of
AAV-rep78 expression plasmid and 24 hours afterwards infected with
the recombinant Baculo-AAV-GPR at an MOI of 1,000. Cells were
maintained without hygromycin for 4 days and then 5.times.10.sup.5
cells were seeded in 22 10-cm dishes in the presence of hygromycin
(100 .mu.g/ml) at serially diluted concentrations. The 22 dishes
were screened for p24gag production by ELISA. Only one dish, in
which cells were seeded at 3.7.times.10.sup.4 cells/dish, released
sufficient p24gag in the supernatant. The dish contained 40
colonies which were all picked-up and screened. Three of them,
scoring positive for p24gag production, were further
characterized.
Lentiviral Vector (LV) Production and Titration
[0062] Pseudo-typed LVs produced from HEK293T cells were obtained
by transient co-transfection of the following plasmids: the
packaging constructs CMV-GPR (3.sup.rd-generation) [or CMV-GPRT
(2.sup.nd-generation)], the VSV-G or RD114-TR envelope constructs,
and the 3.sup.rd-generation SIN-eGFP.sup.24 or the
2.sup.nd-generation PAN-Chim3 transfer vector.sup.21. The ratio of
packaging:envelope:transfer vector was 6.5:3.5:10 .mu.g DNA unless
otherwise indicated. LV from PK-7 clone were generated by
co-transfecting the env-expressing plasmid and the transfer vector.
Transient transfections were performed with either the standard
Ca.sup.++-phosphate method or the Fugene.TM.6 system following the
manufacturer's instruction (Roche Diagnostics Corporation,
Indianapolis, Ind.) obtaining similar results. Supernatants were
harvested 48 hours after transfection and filtered through a
0.45-.mu.m filter. Titer was calculated on SupT1, CEM A3.01,
primary activated peripheral blood mononucleated cells (PBMC) and
umbilical cord blood derived CD34.sup.+ HSC depending on the type
of experiments. Briefly, SupT1 and activated primary mononucleated
cells were transduced by two cycles of spinoculation (1,240.times.g
for 1 hour) in the presence of polybrene (8 .mu.g/ml)
(Sigma-Aldrich, St Louis, Mo.) separated by an overnight resting
phase; CD34.sup.+ HSCs were transduced for 24 hours on
retronectin-coated plates (Takara Bio, Otsu, Japan) without
polybrene. Transduction efficiency was monitored by flow cytometry
analysis (FACS Calibur BD Bioscience, San Jose, Calif.) of eGFP
expression (SIN-eGFP). or .DELTA.LNFGR expression
(P.DELTA.N-Chim3), as described in Porcellini et al., 2009 &
2010.sup.21,22, using the FlowJo software (Tree Star, Inc.,
Ashland, Oreg.) Only transduction values ranging from 5 to 20%
positive cells were used to calculate the titer of each LV
preparation according to the following formula: TU=[number of
cells.times.(% GFP/100)]/vol sup (in ml).
Southern Blot Assay
[0063] Genomic DNA (gDNA) was isolated by the QIAamp Mini kit
(QIAGEN GmbH, Germany) according to manufacturer's instructions.
Baculovirus DNA was extracted from viral particles by the QIAamp
DNA micro kit (QIAGEN). After overnight digestion with the
indicated restriction enzymes 10 .mu.g of gDNA was run on 0.8%
agarose gel, blotted by Southern capillary transfer onto nylon
membranes (Duralon, Stratagene, Tex., USA) and then hybridized to
1.times.10.sup.6 dpm/ml of .sup.32P-random primed labeled either
600-bp CMV or 11-kb GPR cassette.
Analytical PCR Analysis
[0064] PCR analysis for screening of residual integration of the
AAV-Rep78 plasmid into PK-7 cells was performed on 300 ng of
genomic gDNAs using the set of primers: AAV-Rep78 forward: 5'-CGG
GCT GCT GGC CCA CCA GG-3'; AAV-Rep78 reverse: 5'-ATG CCG GGG TTT
TAC GAG ATT GTG-3' and the following PCR conditions: 98.degree. C.
for 7 minutes, 30 cycles of 94.degree. C. for 30 seconds,
66.degree. C. for 30 seconds, and 72.degree. C. for 1.5
minutes.
[0065] To establish the orientation of the two GPR cassettes, PCR
amplification was performed on 300 ng gDNAs using the set of
primers: rev forward: 5'-CTT GAG GAG GTC TTC GTC GC-3'; beta-globin
reverse: 5'-CCC TGT TAC TTC TCC CCT TCC-3'; rev forward nested:
5'-TGT CTC CGC TTC TTC CTG CC-3'; beta-globin nested reverse:
5'-TTA ACC ATA GAA AAG AAG GGG-3' and the following conditions:
94.degree. C. for 2 minutes, 35 cycles of 94.degree. C. for 30
seconds, 52.degree. C. for 30 seconds, and 72.degree. C. for 1.5
minutes.
p24gag ELISA
[0066] Physical LV production was measured in culture supernatants
by the Alliance HIV-1 p24 Antigen ELISA kit (Perkin Elmer Life and
Analytical Sciences, Inc. Waltham, Mass.) following manufacturer's
instructions, with the assumption that 1 pg p24gag corresponds to
1.times.10.sup.4 physical particles.
Western Blot Analysis
[0067] Whole-cell and nuclear extracts derived from PK-7 cells and
viral proteins derived from isolated cell-free VLP or LV were
prepared as previously described.sup.21,22. Proteins were
size-fractionated by SDS-PAGE, and electroblotted to Hybond ECL
nitrocellulose membranes (GE Healthcare, Life Sciences, UK Ltd,
UK). Membranes were blocked in 5% low-fat dry milk, and then
incubated with the appropriate primary Ab. The anti-HIV serum,
obtained from an AIDS patient, was used at 1:2,000 dilution; the
HIV-1 rev MoAb (Rev-6, sc-69730 S. Cruz Biotechnology, Inc., S.
Cruz, Calif.) at 1:200 dilution. Ab binding was visualized by the
enhanced chemiluminescence system ECL (ECL, Amersham) following
manufactures's instructions.
LV DNA Copy Number Quantification by Real-Time TaqMan PCR
[0068] The copy number of the integrated GPR vector was established
by quantitative Tag Man PCR using an ABI Prism 7,900 FAST
instrument (Applied Biosystems, Foster City, Calif.) and analyzed
by SDS 2.3 software (Applied Biosystems). Genomic DNA (gDNA) was
amplified with the following primers and probe derived from the gag
gene: forward: 5'-ACA TCA AGC AGC CAT GCA AAT-3'; reverse: 5'-ATC
TGG CCT GGT GCA ATA GG-3'; probe: FAM 5'-CAT CAA TGA GGA AGC TGC
AGA ATG GGA TAG A-3' TAMRA. PCR conditions were the following: 2
minutes at 50.degree. C. and 5 minutes at 95.degree. C., followed
by 40 cycles of 15 seconds at 95.degree. C. and 15 seconds at
60.degree. C., with an increment of 0.1.degree. C./cycle.
Ligation-Mediated (LM)-PCR
[0069] Genomic DNA was extracted from PK-7 cells by QIAamp DNA Mini
Kit (QIAGEN) according to the manufacturer's instructions and
digested with BgllI and BamHI at 37.degree. C. overnight. Ligation
of an adaptor 76-bp oligonucleotide linker compatible with the
5'-GATC-3' sticky ends was performed under standard conditions.
LM-PCR was carried out using the following couple of nested
primers: the ITR forward: 16 s: 5'-GTA GCA TGG CGG GTT AAT CA-3',
and 17 s/long nested: 5'-TTA ACT ACA AGG AAC CCC TAG TGA TGG-3';
the linker reverse primers: Linker-1: 5'-GTA ATA CGA CTC ACT ATA
GGG C-3' and Linker-2 nested: 5'-AGG GCT CCG CTT AAG GGA C-3'. The
linker sequences corresponded to 5'-GAT CGT CCC TTA AGC GGA GCC CTA
TAG TGA GTC GTA TTA CCA GGG AAT TCG CCT CGG GAT ATC ACT CAG CAT AAT
G-3'. Two rounds of LM-PCR were carried out using AmpliTaq Gold DNA
Polymerase (Applied Biosystems), each comprising 30 cycles
(95.degree. C. for 30 seconds, 52.degree. C. for 30 seconds,
72.degree. C. for 2 minutes). PCR amplicons were cloned using the
TOPO.RTM. cloning kit (Invitrogen, Co.) and plasmid colonies
carrying inserts of approximately 100-200-bp were selected for
sequencing. Sequence homologies were identified by BLAST search,
NCBI.
Fluorescence In Situ Hybridization (FISH)
[0070] Metaphase chromosomes were obtained by treating PK-7 cells
with colchicine (10 .mu.g/ml) (Sigma #C9754) for 2 hours at
37.degree. C. After phosphate buffer saline (PBS) washing, cells
were kept in hypotonic solution (75 mM KCl) for 6 minutes at room
temperature (RT), fixed with 4 washes of methanol/acetic acid (3:1)
and then spread on a clean glass slide. Cytogenetic samples were
denatured in 70% formamide solution for 2 minutes at 72.degree. C.,
dehydrated by cold 70%, 85%, and 95% ethanol consecutive washes and
then air dried. The specific probes were prepared as follows: the
13-kb plasmid DNA containing the GFR cassette was labeled using the
Random Primed DNA Labeling Kit (Roche Applied Science,
Indianapolis, Ind.) with SpectrurnOrange.TM.-dUTP (Vysis, Inc.,
Downers Grove, Ill.), whereas the control 30-kb cosmid DNA
containing the human hax4 gene was labeled using the FISHBright.TM.
Nucleic Acid Labeling kit (Kreatech Biotechnology, Amsterdam, The
Netherlands). Hybridization was performed by incubating 5 ng/.mu.l
of each probe in 250 .mu.l of 50% forrnamide, 2.times.SSC, and 10%
dextran sulfate and 50 ng/.mu.l of human C.sub.0T-1 DNA
hybridization buffer (Invitrogen). Samples were coated with
denatured probes for 10 minutes at 75.degree. C., covered with
Parafilm.RTM.M, and incubated overnight at 37.degree. C. in a moist
chamber. Samples were washed once in 0.4.times.SSC, pH=7 at
72.degree. C. for 2 minutes, once in 4.times.SSC, pH=7 containing
0.0025% Tween-20 for 30 seconds at RT and twice in PBS 1.times. for
1 minute at RT. Slides were counterstained with 0.02 .mu.g/.mu.l of
49,6-diamidino-2-phenylindole (DAPI) (Sigma). Visualization and
photographic images were taken with a Nikon 80i upright microscope
(Nikon Instruments S.p.A., Italy) using the green (FITC) and
spectrum orange (spectrum orange) filter illumination. Images were
processed with Genikon software (Nikon).
Example II
Generation of the First Intermediate PK-7 Clone
[0071] To obtain the RD-Mol Pack packaging cell line for the
continuous production of either 2.sup.nd- or 3.sup.rd-generation
LV, several HEK293T-derived intermediate clones were obtained. The
first one was named PK-7 and was obtained by stable integration of
HIV-1 gag, pol, and rev genes by means of the recombinant hybrid
baculo-AAV vector (rhBaculo-AAV-GPR) (FIG. 1a, scheme 1). This
delivery system exploits the integrase activity of AAV-rep78
protein, provided transiently, to excise and integrate the AAV
ITR-flanked integration cassettes into human chromosomes (FIG. 1b).
The rh-baculo-AAV vector was generated by homologous recombination
between the BaculoDirect Linear DNA and the Gateway.RTM. pENTR.TM.4
entry plasmid containing the ITR-flanked GPR cassettes (FIG. 1a,
scheme 1). After 3 cycles (p3) of recombinant baculovirus
amplification in Sf9 insect cells, the titer and the potential
recombination events of the hybrid baculo-AAV DNA were checked by
plaque assay and viral genomic DNA Southern blot, respectively. The
titer at p3 corresponded to 6.times.10.sup.10 pfu/ml, and Southern
blot analysis revealed a single sharp band, demonstrating no
recombination events during the virus amplification process (FIG.
3).
[0072] Next, the dose and time of AAV-Rep78 plasmid transfection
and of rh-baculo-AAV infection and the cloning conditions of
infected HEK293T cells were carefully defined (FIG. 1c). In fact,
the choice of these experimental settings turned out to be
critical. Thus after testing a wide-range of conditions, it was
eventually established that a single dose of AAV-rep78 plasmid DNA
transfected 24 hours before rh-baculo-AAV infection at the MOI of
1,000 corresponded to the best experimental design. Moreover, it
was observed that seeding a total of 5.times.10.sup.5 cells
distributed in 22 90-mm-Petri dishes, each dish seeded at different
concentration and adding hygromycin 100 .mu.g/ml after 4 days from
seeding was the best condition to collect the largest number of
cell clones. Only three of the 360 counted clones, PK-7, PK-17 and
PK-18, expressed p24gag above the 100 pg/ml settled threshold.
Southern blot analysis of the clones revealed that each clone
contains two copies of the correct-in-size vector (FIG. 2a). To
exclude possible integration of residual AAV-rep78 plasmid DNA,
rep78 specific PCR on PK-7 gDNA were carried out detecting no
positive signal (FIG. 3b). The HIV-1 protein expression pattern
derived from the GPR cassette was monitored by Western blot of the
three PK clones and their matching viral like particles (VLP)
released in the medium. All viral proteins were properly processed,
correct-in-size and in the right, relative proportion (FIG. 2b).
The future working PK clone was selected by calculating on SupT1
cells the potency of the LV produced from the three clones after
being co-transfected with the VSV-G plasmid and the
3.sup.rd-generation transfer vector SIN-eGFP (Table 1). Of note,
although the titer of control HEK293T LV produced by transient
transfection was 5-fold higher than that of PK-7 and PK-18 LV, its
infectivity was almost identical to that of the PK LV, suggesting
that the PK clones generate LV that under a "quality" standpoint
are comparable to those produced by conventional methods (Table 1).
Although the potency of PK-7 and PK-18 LV was similar, PK-7 clone
was selected for further genetic manipulation because its
morphology, growth, viability and p24gag production values scored
better than those of PK-18 clone (Table 1).
TABLE-US-00001 TABLE 1 Potency of VSV-G pseudotyped LV produced
from PK clones Clones Titer (TU/ml).sup.a PK-7.sup.b 1.1 .times.
10.sup.7 PK-17 5.4 .times. 10.sup.6 PK-18 1.0 .times. 10.sup.7
HEK-293T 5.8 .times. 10.sup.7 p24Gag (ng/ml) PK-7 406 PK-17 636
PK-18 326 HEK-293T 1694 Infectivity (TU/ng p24Gag) PK-7 2.7 .times.
10.sup.4 PK-17 8.4 .times. 10.sup.3 PK-18 3.0 .times. 10.sup.4
HEK-293T 3.4 .times. 10.sup.4 Titer was calculated on SupT1 cells 3
days fter transduction. Cells were transfected with he VSV-G and
SIN-eGFP plasmids. Bold indicates the selected clone. indicates
data missing or illegible when filed
[0073] Next, the integration of the ITR-flanked GPR cassette was
characterized in depth in PK-7 clone by quantitative LM-PCR, TaqMan
PCR (FIG. 2c) and FISH techniques (FIG. 2d). To exactly map the
integration site, LM-PCR studies were carried out, which
spotlighted the breakpoint at the chromosome 2, 2q32.1 (FIG. 2c).
This result was confirmed by in situ hybridization with the
specific GPR probe which revealed a single spot into chromosome 2
based on the arm length and centromere position (FIG. 2d). To
confirm this location assignment, the HOX4 probe was used, which is
known to map into chromosome 2q31.2. As HEK293T cells are triploid,
HOX4 was rightly detected in all three chromosomes 2 (FIG. 2d).
Lastly, it was confirmed by quantitative TaqMan PCR that two copies
were integrated and by nested PCR with an appropriate design of the
primers (FIG. 2e) that the two copies were in tandem orientation,
tail-to-head. Tail-to-head orientation is the natural configuration
observed also for the integration of wild type AAV and most rAAV
vector concatamers into the host cell genome.sup.16. Sequence
analysis of the amplicon encompassing the tail-to-head junction
revealed that a 910-bp fragment comprising 303-bp of the 3' CMV
promoter of the first cassette together with both ITRs of the first
and second cassette and the entire 5' CMV promoter of the second
cassette were lost (FIG. 2e, red box). The majority of the
vector-cellular recombination events occurs in fact within the ITR
sequences of the vector. This rearrangement has caused in the PK-7
cells the lack of transcription of the gag-pol gene of the second
cassette and likely the lack of transcription of the rev and hygro
genes of the first cassette. However, it is worth mentioning that
the 285-bp region left of the deleted CMV promoter (FIG. 2e, gray
triangle in the center of the scheme) still contains the TATA box
that might be sufficient to drive transcription of the rev and
hygro genes. In conclusion, PK-7 contains two integrated cassettes,
which collectively transcribe one gag-pol gene and one or two rev
and hygro genes.
[0074] To demonstrate the stability of PK-7 clone over time, the
cells were cultivated in the presence or absence of hygromycin for
350 days, corresponding to ca 420 cell doublings, and measured
p24gag production on a per cell basis (Table 2). The average
production in the presence of hygromycin corresponds to
15.34.+-.8.475D ng p24gag/1.times.10.sup.6 cells, whereas in the
absence of antibiotic is 6.70.+-.3.51SD ng p24gag/1.times.10.sup.6
cells (Table 2).
[0075] This difference likely derives from the fact that hygromycin
drug pressure keeps on an "on" state the transcription of the hygro
resistance gene and thereby the chromatin as well. This might
favour the higher transcription of the gag-pol genes. To evaluate
whether the VLP generated from PK-7 clone were functional even
after hundreds of doublings, PK-7 cells were co-transfected at p60
and p102 with VSV-G envelope and SIN-eGFP transfer vector and
calculated the LV potency on SupT1 cells. Remarkably, the titer and
infectivity of LV produced both in the presence and absence of the
selection drug persisted to normal level still after such prolonged
time (Table 2). These data demonstrate no genetic instability of
the GPR cassette regardless the presence or absence of drug
pressure and allowed us to avoid the use of hygromycin in future
characterization. No comparable data regarding the integration
stability of an AAV-ITR mediated cassette are available in the
literature. The only related information is that a human bone
marrow derived, fibroblast-like cell line (Ruddle's Detroit 6
cells) infected with wild type AAV serotype 2 (AVV-2) maintained
viral sequences in a latent state for at least 47 passages and 118
passages.sup.22,23. Notably, PK-7 cells survived for at least 102
passages.
TABLE-US-00002 TABLE 2 Stability of PK-7 clone over time Hygromycin
No Hygromycin Passage p24Gag ng/10.sup.6a p24Gag ng/10.sup.6a P2
10.00 8.10 P6 7.00 4.80 P10 11.40 4.00 P16 5.00 4.80 P20 9.30 5.20
P24 7.20 6.50 P28 9.20 4.40 P32 6.20 8.80 P36 11.00 9.60 P40 18.00
10.00 P44 4.30 8.40 P48 37.50 3.80 P52 7.00 3.20 P56 11.00 6.70 P60
19.00 4.40 P64 22.00 18.70 P68 15.20 8.00 P72 16.50 4.90 P76 17.80
7.60 P80 30.00 11.00 P84 27.00 8.40 P88 23.20 3.50 P92 23.00 7.50
P98 16.70 1.36 P102 19.00 3.85 Mean .+-. SD 15.34 .+-. 8.47 6.70
.+-. 3.51 Titer (TU/ml).sup.b P60 3.2 .times. 10.sup.6 2.0 .times.
10.sup.6 P102 2.7 .times. 10.sup.6 1.3 .times. 10.sup.6 p24Gag
(ng/ml).sup.b P60 86 38 P102 80 13 Infectivity (TU/ng p24Gag).sup.b
P60 4.2 .times. 10.sup.4 5.2 .times. 10.sup.4 P102 3.3 .times.
10.sup.4 1.0 .times. 10.sup.5 .sup.ap24Gag level expressed as ng/1
.times. 10 cells .sup.bPotency values of VSV-G pseudotyped LV
produced after transfection of PK-7 cells with SIN-eGFP and VSV-G
plasmids and tested on SupT1 cells 3 days after transduction
indicates data missing or illegible when filed
Example III
Use of PK-7 Clone for Semi-Stable LV Production
[0076] To better establish whether the semi-stable LV production
from PK-7 clone was overall comparable to that of transient LV
production from HEK293T cells, both cell types were transfected
with the same amount of the necessary plasmids and measured the
percentage of transfection and the potency of their respective LVs
on different target cells (Table 3). In this condition, the mean of
the percentage of transfection of 11 experiments with HEK293T cells
was 90.54.+-.3.6 SEM and that of 12 experiments with PK-7 cells was
91.+-.5.3SEM, indicating that PK-7 cells maintain their high-level
transfection capability. Then, the LV titer was calculated in SupT1
cells, as our standard reference cell type, cord blood derived
CD34.sup.+HSC, and anti-CD3/IL-2 activated cord blood mononucleated
cells (indicated as T lymph. in Table 3) (Table 3).
TABLE-US-00003 TABLE 3 Potency of VSV-G pseudotyped 3.sup.rd and
2.sup.nd generation LV produced from PK-7 clone calculated on
different target cell types Vector Producer SupT1 CD34 T Lymph.
Titer (TU/ml) SIN-eGFP PK-7 5.7 .times. 10 2.4 .times. 10.sup.4 2.3
.times. 10.sup.5 HEK-293T.sup.b 5.6 .times. 10.sup.7 3.0 .times.
10.sup.5 1.3 .times. 10.sup.6 p24Gag (ng/ml) PK-7 45 45 23 HEK-293T
350 350 279 Infectivity (TU/ng p24Gag) PK-7 1.2 .times. 10.sup.5
5.3 .times. 10.sup.2 1.0 .times. 10.sup.4 HEK-293T 1.6 .times.
10.sup.4 8.6 .times. 10.sup.2 4.6 .times. 10.sup.3 SupT1 CD34 CEM
A3.01 Titer (TU/ml) P.DELTA.N-Chim3 PK-7 2.2 .times. 10.sup.6 1.4
.times. 10.sup.4 3.7 .times. 10 HEK-293T.sup.b 5.8 .times. 10.sup.6
1.6 .times. 10.sup.4 8.8 .times. 10.sup.6 p24Gag (ng/ml) PK-7 27 27
27 HEK-293T 114 114 114 Infectivity (TU/ng p24Gag) PK-7 8.1 .times.
10 5.1 .times. 10.sup.2 1.3 .times. 10.sup.5 HEK-293T 5.0 .times.
10.sup.4 1.4 .times. 10.sup.2 7.7 .times. 10.sup.4 .sup.aVSV-G
pseudotyped LV were produced after transfection of PK-7 cells with
either the SIN-eGFP or the P.DELTA.N-Chim3 and VSV-G envelope
plasmids. .sup.bVSV-G pseudotyped LV were produced after
transfection of HEK-293T cells with CMV-GPR, SIN-eGFP (or
P.DELTA.N-Chim3) and VSV-G envelope plasmids. LV were tested on
target cells after 3-5 days of transduction. indicates data missing
or illegible when filed
[0077] Notably, the infectivity of 3.sup.rd generation LV,
SIN-eGFP, generated from PK-7 cells was ca 1-log lower than and
almost equal to that of control LV when the titer was calculated on
SupT1 and CD34.sup.+ cells, respectively, whereas half-log higher
on activated T lymphocytes. The infectivity of the 2.sup.nd
generation LV, P.DELTA.N-Chim3, generated from PK-7 cells was
almost equal to that of control LV when the titer was calculated on
either SupT1 cells or CD34.sup.+ or CEM A3.01 cells. Collectively,
these findings indicate that PK-7 clone produces LV "qualitatively"
equal to those produced by transiently co-transfected HEK293T
cells. A very important issue to be considered for the application
of PK-7 in semi-stable production of LV is that PK-7 clone did not
lose the ability to be easily transfectable. This feature is quite
commonly lost after genetic modification and cloning of 293 cells
with packaging construct.
[0078] Titration experiments were carried out to establish the dose
of total DNA of the envelope plasmid and transfer vector to be used
for optimal LV production from PK-7 cells. Notably, it was
established that even less than one third of total amount of DNA
(0.3 mg) was sufficient to generate LV with a titer comparable to
that of LV produced with the standard amount of DNA (1 mg) on both
SupT1 and CEM A3.01 cell lines. Therefore using PK-7 for
semi-stable production of LV instead of HEK293T cells will
dramatically cut the high cost of the GMP-grade plasmid DNA.
TABLE-US-00004 TABLE 4 Reduction of total amount of plasmid DNA
(small-scale) 0.3 mg 0.6 mg 1 mg Plasmid DNA SupT1 CEM A3.01 SupT1
CEM A3.01 SupT1 CEM A3.01 Titer 1.2 .times. 10.sup.7 4.9 .times.
10.sup.6 1.1 .times. 10.sup.7 3.5 .times. 10.sup.8 1.0 .times.
10.sup.7 4.1 .times. 10.sup.8 (TU/ml).sup.a p24Gag 111 111 105 105
85 85 (ng/ml) Infectivity 1.0 .times. 10.sup.5 4.4 .times. 10.sup.4
1.0 .times. 10.sup.5 3.3 .times. 10.sup.4 1.1 .times. 10.sup.5 4.8
.times. 10.sup.4 (TU/p24Gag) .sup.aTiter of LVs produced after
transfection of PK-7 cells with P.DELTA.N-eGFP vector and VSV-G
envelope.
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Sequence CWU 1
1
14120DNAartificial sequencessource1..20/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 1cgggctgctg
gcccaccagg 20224DNAartificial sequencessource1..24/mol_type="DNA"
/note="primer" /organism="artificial sequences" 2atgccggggt
tttacgagat tgtg 24320DNAartificial
sequencessource1..20/mol_type="DNA" /note="primer"
/organism="artificial sequences" 3cttgaggagg tcttcgtcgc
20421DNAartificial sequencessource1..21/mol_type="DNA"
/note="primer" /organism="artificial sequences" 4ccctgttact
tctccccttc c 21520DNAartificial sequencessource1..20/mol_type="DNA"
/note="primer" /organism="artificial sequences" 5tgtctccgct
tcttcctgcc 20621DNAartificial sequencessource1..21/mol_type="DNA"
/note="primer" /organism="artificial sequences" 6ttaaccatag
aaaagaaggg g 21721DNAartificial sequencessource1..21/mol_type="DNA"
/note="primer" /organism="artificial sequences" 7acatcaagca
gccatgcaaa t 21820DNAartificial sequencessource1..20/mol_type="DNA"
/note="primer" /organism="artificial sequences" 8atctggcctg
gtgcaatagg 20931DNAartificial sequencessource1..31/mol_type="DNA"
/note="probe" /organism="artificial sequences" 9catcaatgag
gaagctgcag aatgggatag a 311020DNAartificial
sequencessource1..20/mol_type="DNA" /note="primer"
/organism="artificial sequences" 10gtagcatggc gggttaatca
201127DNAartificial sequencessource1..27/mol_type="DNA"
/note="primer" /organism="artificial sequences" 11ttaactacaa
ggaaccccta gtgatgg 271222DNAartificial
sequencessource1..22/mol_type="DNA" /note="primer"
/organism="artificial sequences" 12gtaatacgac tcactatagg gc
221319DNAartificial sequencessource1..19/mol_type="DNA"
/note="primer" /organism="artificial sequences" 13agggctccgc
ttaagggac 191476DNAartificial sequencessource1..76/mol_type="DNA"
/note="linker sequence" /organism="artificial sequences"
14gatcgtccct taagcggagc cctatagtga gtcgtattac cagggaattc gcctcgggat
60atcactcagc ataatg 76
* * * * *